The goal of this work is to define the factors that regulate the in vitro proliferation and differentiation of primitive hematopoietic stem cells. In order to study these stem cells, it will be necessary to isolate them from the confounding influences of colony forming cells and other marrow accessory cells. Our approach is based on our studies using monclonal antibodies to define antigens on in vitro colony forming cells and the precursors of colony forming cells, detected in long term marrow cultures. As a result of these studies we can now separate these precursors from their colony forming progeny (CFU-GM, BFU-E, CFU-Mix). Specifically, we have been able to show that precursors of colony forming cells express the 12.8 (p115) and p4.1 (HLA-DR beta chain) antigens but not the L4F3 (p67) antigen. In contrast, colony forming cells express all of these antigens. As a result, we have been able to isolate 12.8 positive marrow cells, deplete these of L4F3 positive colony forming cells, and show that the remaining cells contain precursors of colony forming cells. Therefore, first, we are developing strategies to purify the precursors of colony forming cells using monoclonal antibodies and cell separation techniques that I have developed. Second, we then will use the purified precursors to develop a clonal assay for these stem cells. Third, we then can use purified precursors of colony forming cells and the clonal assay to define soluble and cellular factors that influence the in vitro proliferation of these stem cells. Fourth, we then can use purified precursors of colony forming cells, purified growth factors, and the colonal assay to determine whether the probability of an individual precursor cell differentiating into a specific type of colony forming cell can be influenced by extrinsic factors.